KR20150111941A - Vaccines with higher carbohydrate antigen density and novel saponin adjuvant - Google Patents

Abstract

The present invention provides a vaccine comprising a carbohydrate antigen conjugated to a diphtheria toxin (DT) as a carrier protein, wherein the ratio of the number of carbohydrate antigen molecules to the number of carrier protein molecules is greater than 5: 1. Methods of inhibiting cancer cells by administering a novel saponin adjuvant and an effective amount of the vaccine described herein are also described herein.

Description

VACCINES WITH HIGHER CARBOHYDRATE ANTIGEN DENSITY AND NOVEL SAPONIN ADJUVANT BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a vaccine having high carbohydrate antigen density and novel saponin adjuvant,

Before and after the related application - References

This application claims the benefit of U.S. Serial No. 61 / 748,880, filed January 4, 2013, the disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Cancer vaccines are designed to treat cancer by increasing the body's natural ability to protect itself through the immune system. This has always presented a very attractive therapeutic approach, especially in view of the many drawbacks of conventional surgery, radiation and chemotherapy in the management of cancer. However, due to the low immunogenicity of cancer carbohydrate antigens and the fact that many synthetic vaccines mainly induce IgM and induce IgG antibodies to a lesser extent, the efficacy of such cancer vaccines is still low. Various approaches have been explored, such as the use of adjuvants, to aid immune recognition and activation.

There is an unmet need to develop effective adjuvant and cancer vaccines with improved immune responses, particularly IgG responses. The present invention provides vaccines and adjuvants for carbohydrate antigens to meet these and other needs.

A brief overview of the invention

In one embodiment, the invention is directed to a carbohydrate antigen or immunogenic fragment thereof; And a toxoid protein, wherein the ratio of carbohydrate antigen to toxoid protein is in the range of 5: 1 to 39: 1, and wherein the ratio represents the number of molecules of carbohydrate antigen to toxoid protein. IgG production of a vaccine with a carbohydrate antigen to toxoid protein ratio in the range of 5: 1 to 39: 1 was found to be higher than that of a vaccine with a carbohydrate antigen to toxoid protein ratio equal to or less than 4: 1.

One embodiment of the present invention provides a separate compound of formula (I): or a pharmaceutically acceptable salt thereof;

In this formula,

R ¹ is selected from? -D-apios or? -D-xylose;

으로부터 선택된다.R < ² > and R < ³ > are H,

.

Another embodiment of the present invention provides a pharmaceutical composition comprising a compound of formula (I): or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In this formula,

R ¹ is selected from? -D-apios or? -D-xylose;

으로부터 선택된다.R < ² > and R < ³ > are H,

.

A third embodiment of the present invention provides a novel saponin adjuvant, OPT-821, comprising 1857 compound V1A, 1857 compound V1B, 1857 compound V2A and 1857 compound V2B.

A fourth embodiment of the present invention is directed to a carbohydrate antigen or immunogenic fragment thereof; And an OPT-821 saponin adjuvant. In one embodiment, the vaccine further comprises a carrier protein. IgG production, antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) activity of a vaccine with OPT-821 saponin adjuvant was more effective than vaccine with no OPT-821 saponin adjuvant A high was found.

The present invention also provides a method of treating cancer, comprising: (i) administering an effective amount of the vaccine described herein to inhibit cancer cells thereby inhibiting cancer cells; and (ii) administering an effective amount of the vaccine described herein to a subject in need thereof, And a method for inducing the reaction.

The present invention also describes a pharmaceutical composition comprising the vaccine described herein and a pharmaceutically acceptable excipient or carrier.

It is to be understood that the description including such terms does not limit the scope of the claims set forth herein or limit the scope or meaning of the following claims. Embodiments of the invention encompassed by this patent are defined by the following claims rather than by the present summary. This Summary is a high-level overview of various aspects of the present invention and introduces some concepts that are additionally described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used separately from determining the scope of the claimed subject matter. These principles are to be understood with reference to the full specification, any or all of the drawings, and the appropriate portions of each claim.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become more apparent upon reading the accompanying drawings and the following detailed description.

Exemplary embodiments of the present invention are described in detail below with reference to the following drawings:
Figure 1A shows the 24 day quantitative anti-Globo H IgG titers for the composition Globo H / KLH / OPT-821 saponin, Globo H / DT / OPT-821 saponin, Globo H / DT / C34 and Globo H / KLH / An example bar graph.
Figure IB is a line graph illustrating the anti-Globo H IgG titers over the 24 day period of the composition of Figure 1A.
Figure 2 shows the effect of G2 vaccine (Globo H / DT (8: 1)) over a 24 day period in mice; G3 vaccine (Globo H / DT (8: 1) / OPT-821); And GL4 vaccine (Globo H / DT (24: 1) / OPT-821) in vivo. 2A illustrates ADCD raw data. Figure 2B illustrates ADCD standardization data. Figure 2C illustrates CDC source data and Figure 2D illustrates CDC standardization data.
FIGS. 3A and 3B show the results of a composition G1 (Globo H / KLH / OPT-821), G2 (Globo H / DT (3: 1) / OPT-821), G3 and G4 8: 1) / OPT-821), G5 (Globo H / DT (8: 1) / C34), G6 (Globo H / KLH / C34) ) And G8 (PBS). ≪ / RTI >
Figure 4 is an assembly of histograms showing IgM and IgG responses at 10 days, 17 days and 24 days of the composition listed in Figure 3: Panel (A) - (C) RTI ID = 0.0 > IgM < / RTI > Panels (D) - (F) illustrate the IgG response of the compositions listed in FIG. 3 at 10 days, 17 days and 24 days, respectively.
Figures 5A-5C are mass spectral images of OPT-821 (including compounds 1989 and 1857).
Figure 6 is a chromatogram LC-UV image of OPT-821.
Figure 7 is an assembly of a chromatogram LC-MS image of OPT-821.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear and ready understanding of the present invention, certain terms are defined herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

&Quot; Effective amount "as used herein refers to the amount of a compound capable of promoting the symptoms of cancer, including, but not limited to, weight loss, pain and tumor mass, which can be detected radiologically, Quot; means the dose of the vaccine or pharmaceutical composition sufficient to reduce the symptoms.

The term "subject" may refer to a vertebrate subject to cancer or a vertebrate subject to need of cancer treatment. The subject includes a warm-blooded animal such as a mammal, such as a primate, and more preferably a human. Non-human primates are also subjects. The term subject is intended to encompass animals such as cats, dogs and the like, domestic animals (eg, cows, horses, pigs, sheep, goats, etc.) and laboratory animals (eg, mice, rabbits, rats, Pigs, etc.). Thus, meristematic applications and medical formulations are contemplated herein.

The term "alkyl ", as used herein, unless otherwise indicated, refers to a straight or branched chain monovalent hydrocarbon radical containing from _{1 to} 20 carbon atoms, such as C ₁ -C ₈ or C ₁ -C ₄ , (Other chain lengths, e.g., 21-30, may also be included in the present invention). Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and t-butyl.

The term "substantially pure" means that, in its natural state, the compounds normally associated with saponin are virtually absent, exhibit a consistent and reproducible chromatographic reaction, elution profile, and biological activity. The term "substantially pure" is not intended to exclude artificial or synthetic mixtures of saponin and other compounds.

All numbers herein are to be understood as modified by "about ".

Vaccine with high carbohydrate ratio

Tumor-associated carbohydrate antigens generally exhibit inadequate immunogenicity. Conjugated carbohydrate antigens with carrier proteins have been employed to enhance the immunogenicity of the carbohydrate antigens. For example, about 700 Globo H molecules are conjugated to a non-toxic keyhole limpet hemocyanin (KLH) protein, an average of about 2 to 4 Globo H molecules are conjugated to the diphtheria toxin (DT) Globo H molecules are conjugated to bovine serum albumin (BSA) and about 6 Globo H molecules are conjugated to tetanus toxoid (Table 1 of U.S. Patent No. 8,268,969).

The present invention relates to a carbohydrate antigen or immunogenic fragment thereof; Wherein the ratio of carbohydrate antigen to toxoid protein is in the range of 5: 1 to 39: 1, and wherein the ratio is the number of molecules of the carbohydrate antigen or of the immunoglobulin molecule of the immunogenic fragment thereof . Such vaccines exhibit better immunogenicity compared to nucleic acids whose carbohydrate antigen molecule to toxoid protein molecule ratio is less than or equal to 4: 1. Other ranges are also encompassed by the present invention and include, for example, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 20: 1, 21: 1, 22: 1, 23: 1, 24: 1, 25: 36: 1, 37: 1, 36: 1, 27: 1, 28: 1, 29: 1, 30: The ratio of the number of molecules of the molecule to the toxoid protein of the 38: 1 or 39: 1 carbohydrate antigen or immunogenic fragment thereof.

In one embodiment, the toxoid protein is a tetanus toxoid (TT) and the ratio of carbohydrate antigen to TT in the carbohydrate-TT vaccine is in the range of 7: 1 to 12: 1.

The present invention relates to a carbohydrate antigen or immunogenic fragment thereof; Wherein the ratio of carbohydrate antigen to DT is in the range of 5: 1 to 39: 1, and wherein the ratio is between the molecular weight of the carbohydrate antigen or immunogenic fragment thereof to the molecular weight of the DT In another embodiment, the ratio of carbohydrate antigen to DT in the carbohydrate-DT vaccine ranges from 8: 1 to 24: 1.

Examples of carbohydrate antigens include, but are not limited to, Globo H, phase-specific embryonic antigen 3 (SSEA3) (also called Gb5), step-specific embryonic antigen 4 (SSEA-4), Gb- (PSA), sTn (c), Tn (c), and Thomsen-Friedenreich antigens (e.g., Lewis antigens such as sLe ^x , Le ^x , sLe ^a , Le ^a , Le ^y , polysaccharide antigens such as PSA, TF (c)), and gangliosides such as GD1, GD2, GD3, fucosyl, GM1, GM1, GM2, GM3, GD1α and GM2. Other carbohydrate antigens include, but are not limited to, α-galactose, α-Man-6-phosphate, α-L-rhamnose, α-GalNAc (Tn), α- NeuAc-OCH2C6H4-p-NHCOOCH2, Fucα1-2Galβ1- (NeuAc2 2-8) 2 polyacids, NeuAc2-6Galb, NeuAcb2-6Gala (STn), Gala1-3Galb1-4GlaNAcb (NeuAca2-8) 3, GalNAcαa-3 (Fucα1-2) Galβ (blood type A), Galα1-3 (Fucα1-2) Galβ (blood type B), 6Gal-HSO3-SiaLex, 6GluNAc-HSO3-SiaLex and α2-6 sialylation diantennary N-glycans. In one embodiment, the carbohydrate antigen is Globo H. "Globo H" was originally derived from the human breast cancer cell line MCF-7 (Fucα1 → 2Galβ1 → 3GalNAcβ1 → 3Galα1 → 4Galβ1 → 4Glcβ1) (Menard S, Tagliabue E, Canevari S, Fossati G, Colnaghi MI. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res , 43 , 1295-300; and Bremer EG, Levery SB, Sonnino S, Ghidoni R, Canevari S, Kannagi R, Hakomori S. (1984) a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland J Biol Chem. , 259 , 14773-7). Globo H is expressed in a variety of epithelial cell tumors such as colon cancer, ovarian cancer, stomach cancer, pancreatic cancer, endometrial cancer, lung cancer, prostate cancer and breast cancer (Menard S et al. Supra ; Bremer EG et al., Supra ; Globo H is commercially available (e. G., Carbosynth, UK) and is prepared by attaching glycosides to ceramides using methods well known in the art, such as by Fossati G, Balsaria A, Sonnino S, Colnaghi MI. Can be synthesized.

The carbohydrate antigen to toxoid protein ratio is greater than 5: 1 or such vaccines are prepared at basic conditions, i.e., at a pH of 8 or greater, 9 or greater, 10 or greater, 11 or greater, or 12 or greater do. The ratio of carbohydrate antigen to toxoid protein can be determined by methods known in the art, for example, MALDI-TOF mass spectrometry. U.S. Patent No. 8,268,969; Also see Morelle W , Faid V , Chirat F , Michalski JC . Methods Mol Biol . 2009; 534: 5-21. doi: 10.1007 / 978-1-59745-022-5_1.Analysis of N- and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry.

The vaccine may further comprise an adjuvant, wherein the adjuvant is a saponin, such as OPT-821 as described herein or a synthetic analog of? -Galactosyl-ceramide (? -GalCer or Cl).

The terms "alpha -galactosyl-ceramide" and "alpha -GalCer ", as used herein, It is a glycolipid that stimulates killer T cells. In one embodiment, the? -GalCer adjuvant has the following structure:

Wherein R is (CH ₂ ) ₂₄ CH ₃ , (CH ₂ ) ₇ PhF, (CH ₂ ) ₁₀ PhOPhF or (CH ₂ ) ₁₀ PhF.

In one embodiment, R is (CH ₂ ) ₁₀ PhOPhF, known as a C34 adjuvant having the structure:

.

.

New  Saponin Adjuvant

The present invention provides a substantially pure OPT-821 saponin. The present invention includes substantially pure OPT-821 saponins as well as biologically active fragments. The present invention may also include an impure form of OPT-821 saponin. The purified OPT-821 saponin exhibits an enhanced adjuvant effect when administered with the vaccine described herein or mixed with other substantially pure saponin or non-saponin adjuvants.

OPT-821 saponin can be prepared, for example, by high pressure liquid chromatography (HPLC), low pressure liquid silica chromatography, and hydrophilic reciprocal chromatography as described in U.S. Patent 5,057,540 and U.S. Patent 6,524,584, (HILIC) by Quillaja Saponaria Molina saponaria Molina) is a naturally occurring glycoside which is extracted from the bark of trees by a high purity system. High pressure liquid chromatography analysis indicates that OPT-821 is a mixture of structurally related isomeric compounds. Purified different isomeric compounds of OPT-821 saponin have been identified and described herein.

OPT-821 saponin comprises at least one isolated compound of formula (I) as follows:

In this formula,

R ¹ is? -D-apios or? -D-xylose;

R ² and R ³ are independently H, alkyl,

(1989 화합물에 대한 지방산 아실 모이어티), 또는

(Fatty acid acyl moiety for the 1989 compound), or

(1857 화합물에 대한 지방산 아실 모이어티)이다.

(Fatty acid acyl moiety for the 1857 compound).

OPT-821 saponin may also comprise a separate compound of formula I wherein (i) R ¹ is? -D-apiose, R ² is a fatty acid acyl moiety for the 1989 compound depicted above and R ³ is H (1989 compound V1A); (ii) R ¹ is? -D-apiose, R ² is H and R ³ is a fatty acid acyl moiety for the 1989 compound described above (1989 compound V1B); (iii) R ¹ is? -D-xylose, R ² is a fatty acid acyl moiety for the 1989 compound depicted above and R ³ is H (1989 compound V2A); (iv) R ¹ is? -D-xylose, R ² is H, and R ² is a fatty acid acyl moiety for the 1989 compound described above (1989 compound V2B). In general, 1989 compound V1A, 1989 compound V1B, 1989 compound V2A and 1989 compound V2B are referred to as " 1989 compound mixture ".

Table 1 summarizes the functional groups of the 1989 compound and the mole percent of each 1989 compound in the 1989 compound mixture.

Table 1

OPT-821 saponin may comprise a separate compound of formula (I) wherein (i) R ¹ is? -D-apiose, R ² is a fatty acid acyl moiety for the 1857 compound depicted above and R ³ is H (1857 compound V1A); (ii) R ¹ is? -D-apiose, R ² is H and R ³ is a fatty acid acyl moiety for the 1857 compound described above (1857 compound V1B); (iii) R ¹ is β-D-xylose, R ² is a fatty acid acyl moiety for the 1857 compound described above, and R ³ is H (1857 compound V2A); (iv) R ¹ is β-D-xylose, R ² is H, and R ³ is a fatty acid acyl moiety for the 1857 compound described above (1857 compound V2B). In general, 1857 compounds V1A, 1857 compounds V1B, 1857 compounds V2A and 1857 compounds V2B are referred to as "1857 compound mixture ".

Table 2 summarizes the mole percent of each 1857 compound in the 1857 compound mixture and 1857 compound mixture.

Table 2

OPT-821 saponin comprises one or more of the following compounds: (i) 1857 Compound VIA; (ii) 1857 compound V1B; (iii) 1857 compound V2A; (iii) 1857 Compound V2B; (iv) 1989 compound V1A; (v) 1989 Compound V1B; (vi) 1989 Compound V2A; Or (vii) 1989 Compound V2B. The percentage of the 1857 compound mixture and the 1989 compound mixture in OPT-821 saponin may be in the following ranges:

(i) from about 1 mol% to about 15 mol% of OPT-821 is comprised of a mixture of 1857 compounds; And

(ii) from about 85 mol% to about 99 mol% of the OPT-821 is made up of a mixture of 1989 compounds.

All mole% can be varied by 0.1% increments (e.g., from about 87% to about 90%, from about 90.5% to about 97%, from about 3.5% to about 11%, from about 10% to about 14%).

1989 The compound mixture comprises about 60-70 mole% of the 1989 compound V1A; About 1-5 mole% of the 1989 compound V1B; About 30-40 mole% of the 1989 compound V2A; And about 0.1-3 mole% of the 1989 compound V2B. All mol% can be varied by 0.1% increments (e.g., 65%, 2.5%, 35.6%).

1857 compound mixture contains about 60-70 mole% of 1857 compound V1A; About 1-5 mole% of 1857 compound V1B; About 30-40 mole percent of 1857 compound V2A; And about 0.1-3 mole% of 1857 compound V2B. All mole% can be varied by 0.1% increments (e.g., 67%, 1.5%, 33.9%).

In another embodiment, the substantially pure OPT-821 is purified from crude quillazana saponaria extract and the OPT-821 has a concentration of A: B 95%: 5% to 75%: 25% , A mobile phase A was distilled water with 0.1% trifluoroacetic acid and mobile phase B was acetonitrile with 0.1% trifluoroacetic acid at a flow rate of 1 ml / min. When analyzed for reversed phase-HPLC on a Symmetry C18 column with 4.6 mm ID x 25 cm L, characterized by a single dominant peak comprising 90% or more of the total area of all peaks in the chromatogram, excluding the solvent peak .

In one embodiment, the pharmaceutical composition comprises a compound of formula (I): < EMI ID =

In this formula,

R ¹ is? -D-apios or? -D-xylose;

R ² and R ³ are independently H, alkyl, or

(1857 화합물에 대한 지방산 아실 모이어티)이다.

(Fatty acid acyl moiety for the 1857 compound).

The vaccine may comprise a carbohydrate antigen or immunogenic fragment thereof and OPT-821 saponin. In another embodiment, the vaccine comprises a carbohydrate antigen, DT, and OPT-821 saponin selected from Globo H, SSEA-3, SSEA-4, Gb-4 or a mixture thereof. In yet another embodiment, the vaccine comprises a carbohydrate antigen or an immunogenic fragment thereof; Carrier protein and OPT-821 saponin. Non-limiting examples of carrier proteins include toxoid proteins and non-toxoid proteins such as KLH.

Toxoid  protein

The toxoid protein conjugated to a carbohydrate antigen may be a diphtheria toxin (DT) or a tetanus toxoid (TT).

The toxin may be inactivated by treatment with, for example, formaldehyde, glutaraldehyde, UDP-dialdehyde, peroxides, oxygen, or by mutation (e.g., using recombinant methods). Newell < / RTI > Vaccines, Marcel Dekker, Inc., New York, 1989. Genth et al ., Inf. and Immun., 68 (3): 1094-1101,2000). A mutant primary diphtheria toxin with reduced toxicity can also be generated using recombinant methods. U.S. Patent 5,085,862; 5,221,618; 5,244,657; 5,332,583; 5,358,868; And 5,433,945.

DT is the diphtheria toxin cross-reactant (DT-CRM) or diphtheria toxoid. DT-CRM means a mutant primary diphtheria toxin that no longer has sufficient ADP-ribosyl, for example, by mutation or by chemical modification. Non-limiting examples of DT-CRM include DT-CRM 30, DT-CRM 45, DT-CRM 176, DT-CRM 197 and DT-CRM 228. Diphtheria toxoid is a formaldehyde-inactivated diphtheria toxin. DT may be manufactured by methods known in the art, such as the recombinant DNA technique described in U.S. Patent No. 5,614,382, which is commercially available or is incorporated by reference in its entirety.

Carbohydrate antigens of the vaccines described herein may be covalently bound to a carrier protein via a p-nitrophenyl linker by the synthetic process described in U.S. Patent No. 8,268,969, the entire contents of which are incorporated by reference.

The vaccine of the invention can elicit one or more of the following activities: high IgG titers, high complement-dependent cytotoxicity (CDC) activity, and / or high antibody-dependent cell-mediated cytotoxicity (ADCC) activation. In another embodiment, the vaccine induces one or more of the following cells: a natural killer cell, a CD4 + T lymphocyte or a CD8 + T lymphocyte. Other immunological variables, including but not limited to T helper cell activation, can be measured.

The present invention also provides a pharmaceutical composition comprising the vaccine described herein and a pharmaceutically acceptable vehicle, excipient or carrier. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the vehicle may contain wetting or emulsifying agents, pH buffering agents, or other excipients such as adjuvants. Pharmaceutically acceptable carriers may contain physiologically acceptable compounds which act on, for example, stabilize, increase or decrease the absorption or removal rate of the pharmaceutical compositions of the present invention. Physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, detergents, liposome carriers or other stabilizers and / Or buffers. Excipients can be nonionic surfactants, polyvinylpyrrolidone, human serum albumin, aluminum hydroxide, anesthetic agents, and a variety of unmodified derivatized cyclodextrins. More preferably, the nonionic surfactant may comprise polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. The polyvinylpyrrolidone may be Plasdone C15, preferably a pharmaceutical grade polyvinylpyrrolidone. The anesthetic agent is preferably benzyl alcohol. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives. For example, the 21st edition of Remington ' s Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ("Remington's"). The pharmaceutical compositions of the present invention may also contain adjuvants such as pharmacological agents, cytokines, or other biological response modifiers. Pharmaceutical compositions comprising such excipients or carriers are formulated by any of the well-known conventional methods.

The vaccine may be formulated for the following routes of administration: intramuscular, intradermal, oral, transdermal, nasal, oral, rectal, vaginal, by inhalation, or by subcutaneous administration. Other modes of administration may be applied as long as satisfactory immunogenicity can be induced.

The pharmaceutical compositions of the present invention may be prepared injectable, either as a liquid solution or suspension, or in solid form suitable for being dissolved or suspended in a liquid vehicle prior to injection. The pharmaceutical compositions may also be prepared as an active ingredient encapsulated in a liposomal vehicle or other particulate carrier for use in emulsified solid forms or sustained delivery. For example, the pharmaceutical composition may be in the form of an oil-in-water emulsion, a water-in-oil emulsion, an oil-in-water emulsion, a site-specific emulsion, an organ- , Nanoparticles and various natural or synthetic polymers such as non-resorbable impermeable polymers such as ethylene vinyl acetate copolymers and Hytrel copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or Polyesters, such as those used to make resorbable sutures that allow for sustained release of the vaccine.

The pharmaceutically acceptable salts and the physiologically functional derivatives of the compounds of the present invention can be prepared by reacting an alkali metal (e.g. sodium, potassium), an alkaline earth metal (e.g. calcium, magnesium), ammonium and NX ₄ ⁺ , And X is C ₁ -C ₄ alkyl). Pharmaceutically acceptable salts of amino groups include organic carboxylic acids such as tartaric acid such as formic acid, glucuronic acid, malic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, (Methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, toluenesulfonic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, stearic acid, maleic acid, maleic acid, Aliphatic, cycloaliphatic, aromatic, heterocyclic, carboxylic acid, and esters of an organic acid such as lactobionic acid, fumaric acid, and succinic acid, such as, for example, Sulfonic acid class; Organic sulfonic acids such as methaniesulfolic acid, ethanesulfonic acid, isothionic acid, benzenylesulfonic acid and p-toluenesulfonic acid; And salts of inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carboxylic acid, sulfuric acid, sulfamic acid and phosphoric acid. Pharmaceutically acceptable salts of compounds having a hydroxy group include Na ⁺ , NH ₄ ⁺ or NX ₄ ⁺ (where X is, for example, a C ₁ -C ₄ alkyl group), Ca ⁺⁺ , Li ⁺ , Mg ⁺⁺ , or an anion of the compound together with a suitable cation such as K ^{& lt} ; ⁺ & gt ^; and zinc, or an organic salt thereof may be a primary, secondary and tertiary amine, cyclic amine, N, N'- dibenzylethylenediamine, chloroprocaine, choline , Diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All such salts can be prepared from the corresponding compounds by conventional means, for example by reacting the appropriate acid or base with the free form of the compound.

Inducing immune response / Cancer cells  How to suppress

Another aspect of the invention is directed to a method of inducing an immune response comprising administering an effective amount of a vaccine described herein to a subject in need thereof. Immune responses include, but are not limited to, NK cell response, ADCC and CDC activity, and IgM and IgG production.

In yet another aspect, the invention provides a method of inhibiting cancer cells, comprising administering an effective amount of the vaccine described herein to a subject in need thereof. In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, esophageal cancer, rectal cancer, biliary cancer, liver cancer, bowel cancer, stomach cancer, colon cancer, parietal cancer, kidney / kidney cancer, prostate cancer, ovarian cancer, cervical cancer, Neoplasms, cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine cancer, adrenocortical cancer, thyroid cancer, bone cancer, skin cancer such as basal cell carcinoma, squamous cell carcinoma or melanoma. In another embodiment, the cancer is a Globo H-expressing cancer. Non-limiting examples of Globo H expression cancers include breast cancer, lung cancer, gastric cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, and endometrial cancer. Antibodies produced by vaccines, such as anti-Globo H antibodies, essentially inhibit Globo H-expressing cancers.

In certain embodiments, an effective amount of a vaccine is one that elicits a desired immunological effect, such as stimulating IgG production on a particular carbohydrate antigen (e. G., Globo H) in a subject. The effective amount or dose of the vaccine or pharmaceutical composition may vary depending on the amount of carbohydrate antigen, the type of adjuvant used, the mode of administration, and the age, size, and condition of the subject being treated. The exact amount of vaccine or pharmaceutical composition required to induce immunogenicity will be determined by the attending physician.

The vaccine may be administered at a stat dose with or without one or more additional doses at specific time intervals to achieve long term immunoprotective effects from months to years. The frequency of administration may depend on any of a variety of factors such as the severity of the symptoms, the severity of the desired immune protection, whether the pharmaceutical composition has been used for prophylactic or therapeutic purposes, and the like. For example, in one embodiment, the pharmaceutical composition of the present invention is administered once a month, twice a month, three times a month, every other week, qw every week, biw every week, three times a week, (Qid), twice daily (qd), twice daily (qid), or three times a day (tid) daily, twice a week. The vaccine may also be administered concurrently or sequentially with other conventional therapies, such as antibodies that target chemotherapy, targeted therapies, or tumor-associated carbohydrate antigens for cancer therapy.

The present invention is further illustrated by the following examples, which are provided for purposes of illustration rather than limitation. It should be understood by those skilled in the art that many changes may be made in the specific embodiments described and that the same or similar results may still be obtained without departing from the spirit and scope of the invention.

Example  1: Preparation of vaccine with higher carbohydrate / toxin protein ratio and extraction of OPT-821

Globo H has been conjugated to KLH or DT according to methods known in the art, for example, as described in U. S. Patent No. 6,544, 952 or 8,268, 969, the entire contents of which are incorporated herein by reference. The resulting vaccine contained Globo H: DT (ratio of molecules of Globo H to DT = 2-4: 1).

General procedure for generating glycoconjugates

The glycoconjugate was prepared as follows:

BSA, DT-CRM197, and tetanus toxoid (Adimmune, Taiwan) were dissolved in 100 mM phosphate buffer pH 7.2 (~ 5 mg / ml) and 30 to 40 equivalents of Globo H half ester 35 was added to the solution. The mixture was gently stirred at room temperature for 24 hours. The mixture was then diluted in deionized water and dialyzed against 5 changes of deionized water. The solution was then lyophophilized with a white powder. The resulting Globo H-protein conjugate can be characterized by MALDI-TOF analysis to determine the carbohydrate incorporation rate. 41 (GH-BSA), MALDI-TOF data 76029, 42 (GH-DT-CRM 197) data 62138 and 43 (GH-TT) data 162902 and 44 (GH-BaMV). MALDI-TOF MS analysis of glycoconjugates. The glycoconjugate and primary carrier protein can be reconstituted with ddH ₂ O (~ 1 μg / μl). The matrix cininic acid was freshly prepared with 1: 1 acetonitrile and deionized water to a final matrix concentration of 10 mg / ml containing 0.1% TFA. The matrix solution and glycoconjugate were gently loaded and mixed, and the plate was air dried. Calibration using bovine serum albumin was essential before measurement. Each glycoconjugate and primary protein sample was detected under linear positive mode. The average molecular weight makes it possible to calculate the average number of carbohydrate molecules incorporated on the carrier protein.

A vaccine with a carbohydrate antigen molecule: toxin protein mole ratio of greater than 5: 1 was prepared according to the following steps:

(a) 10 ml-25 ml of Globo H (available from OBI Pharma, Taiwan) and p-nitrophenyl ester linker (commercially available from OBI Pharma, Taiwan) were dissolved in 25 μl DMF (Sigma-Aldrich, USA) .

(b) 25 mg of DT was dissolved in 2.5 ml of phosphate buffer (i.e., a basic buffer with pH> 8).

(c) The mixture of step (a) was added to the mixture of step (g) at room temperature overnight. The resulting mixture had a pH of 8 to 9.2.

Results: 10 ml of Globo H produced a vaccine containing Globo H: DT (8: 1) and 25 Globo H contained Globo H: DT (24: 1) as determined by MALDI-TOF MS Lt; / RTI >

Preparation of OPT-821 Saponin

OPT-821 saponin was extracted from the quillaja saponaria extract according to the following steps:

(a) Quillaja saponaria extract was pre-filtered by large particle C18 reverse phase chromatography and then purified by silica-based preparative normal chromatography. This resulted in crude OPT-821.

(b) crude OPT-821 of step (a) was again pre-filtered by large particle C18 reverse phase chromatography and then treated with reverse phase preparative HPLC. OPT-821 material was sequenced sequentially by desalting and freeze drying.

Purified OPT-821 saponin extracted from the bark of Quillaja saponaria molina tree was analyzed by mass spectrometry. The mass peak of 1989.01 in FIG. 5A, the mass peak of 1989.12 in FIG. 5B, and the mass peak of 1989.13 illustrate the presence of a compound having a molecular weight of about 1989. The molar ratio of the compound having a molecular weight of about 1989 is 89.8% in Figure 5a, 96.8% in Figure 5b, and 87.0% in Figure 5c. Similarly, the mass peaks at 1856.97 in FIG. 5A, the mass peaks at 1856.02 in FIG. 5B and the mass peaks at 1857.09 illustrate the presence of a compound having a molecular weight of about 1857. The molar ratio of the compound having a molecular weight of about 1857 is 10.2% in Fig. 5A, 3.2% in Fig. 5B, and 13% in Fig.

The purified OPT-821 saponin was further analyzed by chromatography. Figure 6 is a chromatogram LC-UV image (column: PolyLC PolyHYDROXYETHYL A 200 * 4.6 mm 5 um, 300 A). The first peak represents the presence of the 1989 V1 (A & B) compound and the 1857 compound V1 (A & B) compound (about 65.94%), the second peak represents the presence of the 1989 V2 (A & B) compound (about 34.06%). Figure 7 is a chromatogram LC-MS image (column: Waters Symmetry ODS 150 * 2.1 mm). Peak 1 in the top panel shows the presence of 1989 compounds V1B and V2B (about 2.2%) while peak 4 shows the presence of 1989 compounds V1A and V2A (about 97.8%). Peak 2 of the lower panel represents the presence of 1857 compound V1 B and 1857 compound V2 B (approximately 1.9%) and peak 3 represents the presence of 1857 compound V1A and 1857 compound V2A.

Example  2: immunogenicity of vaccine with higher carbohydrate / toxin protein ratio and adjuvant efficiency of OPT-281 saponin

Evaluation of the in vivo immunogenicity of the Globo H / DT (8: 1) vaccine of Example 1 and the evaluation of the adjuvant efficiency of OPT-821 saponin was performed using CL57B / 6 mice.

About 8 weeks of CL57B / 6 mice were randomized into the following four study groups:

Blood samples were collected either via the eye-back or facial vein without anticoagulant before the first injection or 0 day, and 3 days after each injection (i.e., 10, 17 and 24 days). Blood samples were centrifuged to separate serum and blood cells. Serum was collected and stored at -20 [deg.] C, which was subsequently analyzed by ELISA. Serum from each mouse was serially diluted for anti-Glucose H IgG analysis. Globo H-ceramide was coated overnight on a black plate, blocked with 1X blocking buffer (Sigma) for 30 minutes and washed with PBST. Diluted serum samples were added to the assay plate, incubated for 1 hour at room temperature (RT), and washed. A goat anti-mouse IgG-AP secondary antibody (Southern Biotech) was added to the sample and incubated at RT for 45 minutes. The plate was washed again, then the chromogen substrate was added and incubated at 37 [deg.] C for 20 minutes. The reaction was terminated by adding a stop solution. The optical density was quantified by a plate reader (Molecular Device) at a wavelength of 405 nm. Mann-Whitney t-test was used for statistical analysis. Figures 1A and 1B show the quantitative anti-Globo H IgG titers of the tested vaccine.

Results: IgG reversal from Globo H / DT (non-8: 1) -immunized mice was significantly higher than that of Globo H / KLH using C34 adjuvant (P <0.01). The IgG reversal from the Globo H / DT (non-8: 1) -immunized mouse was higher than that of Globo H / KLH using the OPT-821 saponin adjuvant (see FIG. Regardless of the type of carrier protein used, OPT-821 saponin resulted in statistically significantly higher IgG titers than the C34 adjuvant (P < 0.05, Figs. 1A and 1B).

Example  3: ADCC  And CDC  Assay for the immunogenicity of vaccines with higher carbohydrate / toxin protein ratios and the adjuvant efficiency of OPT-281 saponin

Four groups of Lewis rats were immunized with the vaccine of Table 3.

Table 3: Vaccine composition

Rats were immunized with the vaccines listed in Table 3 on sc, 0, 7, 14, and 21 days. Immunized. Peripheral blood mononuclear cells (PBMC) and plasma were collected before the first injection (i.e., day 0) and at days 10, 17, and 24.

ADCC and CDC assays were performed using the Carlinsin AM release method known in the art. The procedure is described as follows:

Carl Saine  Target cells labeled with AM

MCF-7 breast cancer cells (target cells) were cultured in a minimal essential medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 0.01 mg / mL insulin, 10% fetal bovine serum. Target cells were added to 96-well plates (5 x 10 ³ cells per well) and incubated overnight at 37 ° C in a humidified 5% CO ₂ atmosphere. The medium was discarded and each well was washed once with PBS. 100 L of 20 M calcein-AM solution was added to each well (2 nmole per well) and incubated for 2 hours in a humidified 5% CO ₂ atmosphere at 37 ° C. The supernatant was dried and each well was washed three times with PBS.

Sample Plasma and Incubated  Target cell

Sample plasma was heat-inactivated and 50 L of 1 / 5X heat-inactivated sample plasma was added to each well except "total release" and "background" controls. The final dilution factor after addition of 50 L of PBMC or serum will be 1 / 10X. Plates were incubated at 37 째 C (in a dark room) for 30 minutes.

PBMC  or Complement and Incubated  Target cell

After incubation, 50 microliters of PBMC (2x10 ⁶ cells / mL) (for E: T ratio: 20: 1) were added to each well in an ADCC assay and 50 microliters of 1 / 10X diluted serum was added to CDC Was added to each well except "total release" and "background" controls in the assay. The mixture of reactions was incubated for 4 hours in a humidified 5% CO ₂ atmosphere at 37 ° C. Phenol-red-free MEM (50 microliters) containing 2% Triton solution was added to the "total release" control at an incubation time of at least 15 minutes, phenol- Lt; / RTI &gt; The plate was centrifuged at 100 g for 5 minutes and then 80 microliters of supernatant was transferred to a 96-well black plate. Fluorescence was measured at 485 nm excitation and 538 nm emission wavelength.

Figures 2A-2D show in vivo ADCC and CDC activity of G2, G3 and G4 vaccines.

Results: ADCC and CDC activity of G3 vaccine (having OPT-821 saponin adjuvant) was higher than G2 vaccine (without saponin adjuvant) at 24 days as illustrated in Figures 2B and 2D. As illustrated in Figures 2B and 2D, the ADCD and CDC activity of G4 vaccine (Globo H / DT ratio is 24: 1) at 24 days was higher than that of G3 vaccine (Golbo H / DT ratio was 8: 1). These results indicate that a vaccine with an OPT-821 saponin adjuvant and a carbohydrate antigen / toxin protein ratio of greater than 5: 1 improves ADCC and CDC responses and induces longer lasting ADCC and CDC responses.

Example  4: Immune response of vaccine with higher carbohydrate / toxin protein ratio and adjuvant efficiency of OPT-821 saponin

In vivo evaluation of Globo H / DT (8: 1) and Globo H / DT (16: 1) vaccine and OPT-821 saponin adjuvant in Example 1 was performed using CL57B / 6 or Balb / c mice .

Approximately 8 weeks of CL57B / 6 mice were randomized into the following 8 study groups:

Blood samples were collected either before the first injection or on the 0, 10, 17, and 24 days via the eye-back or facial vein without anticoagulant. Blood samples were centrifuged to separate serum and blood cells. Serum was collected and stored at -20 [deg.] C, which was subsequently analyzed by ELISA. Serum from each mouse was serially diluted for anti-Globo H IgG and IgM analysis. Figures 3A, 3B and 4 show the quantitative anti-Globo H IgM and anti-Globo H IgG titers of the tested vaccine.

Results: The vaccine with OPT-821 saponin adjuvant induced statistically significantly more anti-Globo H IgM and anti-Globo H IgG as compared to the vaccine with C34 adjuvant (Figures 3A, 3B and 4) ). The following statistically significant differences were observed:

G3 백신 (OPT-821 사포닌)의 IgM 역가는 17일에 G5 백신 (C34)의 역가보다 현저하게 더 높았다 (p=0.02);

The IgM potency of G3 vaccine (OPT-821 saponin) was significantly higher than that of G5 vaccine (C34) at 17 days (p = 0.02);

G1 백신 (OPT-821 사포닌)의 IgM 역가는 17일에 G6 백신 (C34)의 역가보다 현저하게 더 높았다 (p = 0.03),

The IgM potency of G1 vaccine (OPT-821 saponin) was significantly higher than that of G6 vaccine (C34) at 17 days (p = 0.03)

G3 백신 (OPT-821 사포닌)의 IgM 역가는 24일에 G5 백신 (C34)의 역가보다 현저하게 더 높았다 (p=0.03),

The IgM potency of G3 vaccine (OPT-821 saponin) was significantly higher than that of G5 vaccine (C34) at 24 days (p = 0.03)

G3 백신 (OPT-821 사포닌)의 IgG 역가는 17일에 G5 백신 (C34)의 역가보다 현저하게 더 높았다 (p=0.001),

The IgG potency of G3 vaccine (OPT-821 saponin) was significantly higher than that of G5 vaccine (C34) at 17 days (p = 0.001)

G1 백신 (OPT-821 사포닌)의 IgG 역가는 17일에 G6 백신 (C34)의 역가보다 현저하게 더 높았다 (p=0.003),

The IgG potency of G1 vaccine (OPT-821 saponin) was significantly higher than that of G6 vaccine (C34) at 17 days (p = 0.003)

G3 백신 (OPT-821 사포닌)의 IgG 역가는 24일에 G5 백신 (C34)의 역가보다 현저하게 더 높았다 (p=0.03), 그리고

The IgG potency of G3 vaccine (OPT-821 saponin) was significantly higher than that of G5 vaccine (C34) at 24 days (p = 0.03), and

G1 백신 (OPT-821 사포닌)의 IgG 역가는 24일에 G6 백신 (C34)의 역가보다 현저하게 더 높았다 (p=0.004).

The IgG potency of G1 vaccine (OPT-821 saponin) was significantly higher than that of G6 vaccine (C34) at 24 days (p = 0.004).

These results illustrate that the OPT-821 saponin adjuvant significantly improves IgM and IgG responses compared to the C34 adjuvant.

Globo H / KLH / OPT-821 saponin (G1) induces significantly higher IgM and IgG titers at 17 and 24 days compared to Globo H / DT (3: 1) / OPT-821 saponin (G2). G1 has a higher carbohydrate density (about 700 Glob H units for the KLH carrier protein) and induces a stronger immune response, while G2 has a lower carbohydrate density (3 for the DT carrier protein) Globo H units) and are thought to induce a weaker immune response. The following statistically significant differences were observed:

G1 백신 (KLH)의 IgM 역가는 17일에 G2 백신 (DT)의 역가보다 현저하게 더 높았다 (p=0.003),

The IgM potency of G1 vaccine (KLH) was significantly higher than that of G2 vaccine (DT) on day 17 (p = 0.003)

G1 백신 (KLH)의 IgM 역가는 24일에 G2 백신 (DT)의 역가보다 현저하게 더 높았다 (p=0.03),

The IgM level of G1 vaccine (KLH) was significantly higher than that of G2 vaccine (DT) at 24 days (p = 0.03)

G1 백신 (KLH)의 IgG 역가는 24일에 G2 백신 (DT)의 역가보다 현저하게 더 높았다 (p=0.004).

The IgG potency of G1 vaccine (KLH) was significantly higher than that of G2 vaccine (DT) at 24 days (p = 0.004).

OPT-821 Saponin (G3 and G4) and Globo H / DT (16: 1 - Globo H to DT molecular ratio) / OPT-821 Saponin (G7) is comparable to IgM and IgG titers of Globo H / KLH / OPT-821 saponin (G1) at 17 and 24 days (see FIG. 4). Despite a lower carbohydrate density than GH-KLH (700: 1 - Globo H vs. DT), GH-DT (molecular ratio of 8: 1 - Globo H vs. DT) has immunogenicity comparable to GH-KLH Respectively.

Vaccines with higher Globo H / DT ratios (8: 1 or 16: 1 - Globo H vs. DT molecular ratio) are higher and longer lasting than vaccines with lower Globo H / DT ratio (3: 1) IgM and IgG titers. The following statistically significant differences were observed:

G3 백신 (8:1 비 - Globo H 대 DT의 분자 비)의 IgM 역가는 17일에 G2 백신 (3:1 비)의 역가보다 현저하게 더 높았다 (p=0.02),

The IgM potency of the G3 vaccine (molecular ratio of 8: 1 non-Globo H vs. DT) was significantly higher (p = 0.02) than that of the G2 vaccine (3: 1 ratio)

G7 백신 (16:1 비 - Globo H 대 DT의 분자 비)의 IgM 역가는 17일에 G2 백신 (3:1 비)의 역가보다 현저하게 더 높았다 (p=0.006),

The IgM potency of the G7 vaccine (molecular ratio of 16: 1 non-Globo H vs. DT) was significantly higher than that of the G2 vaccine (3: 1 ratio) at 17 days (p = 0.006)

G3 백신 (8:1 비 - Globo H 대 DT의 분자 비)의 IgG 역가는 17일에 G2 백신 (3:1 비)의 역가보다 현저하게 더 높았다 (p=0.01),

The IgG potency of the G3 vaccine (molar ratio of 8: 1 non-Globo H vs. DT) was significantly higher than that of the G2 vaccine (3: 1 ratio) on day 17 (p = 0.01)

G7 백신 (16:1 비 - Globo H 대 DT의 분자 비)의 IgG 역가는 17일에 G2 백신 (3:1 비 - Globo H 대 DT의 분자 비)의 역가보다 현저하게 더 높았다 (p=0.03),

The IgG potency of G7 vaccine (molecular ratio of 16: 1 non-Globo H vs. DT) was significantly higher than that of G2 vaccine (molecular ratio of 3: 1 non-Globo H to DT) on day 17 (p = 0.03 ),

G3 백신 (8:1 비 - Globo H 대 DT의 분자 비)의 IgG 역가는 24일에 G2 백신 (3:1 비)의 역가보다 현저하게 더 높았다 (p=0.01),

The IgG potency of the G3 vaccine (molecular ratio of 8: 1 non-Globo H vs. DT) was significantly higher than that of the G2 vaccine (3: 1 ratio) on day 24 (p = 0.01)

G7 백신 (16:1 비 - Globo H 대 DT의 분자 비)의 IgG 역가는 24일에 G2 백신 (3:1 비 - Globo H 대 DT의 분자 비)의 역가보다 현저하게 더 높았다 (p=0.01),

The IgG reversal of G7 vaccine (molecular ratio of 16: 1 non-Globo H vs. DT) was significantly higher than that of G2 vaccine (3: 1 non-Globo H vs. DT) on day 24 (p = 0.01 ),

The IgG titers of OPT-821 saponin (G3) and Globo H / DT (16: 1) / OPT-821 saponin (G7) On the 25th day, the IgG activity of Globo H / DT (3: 1 - Globo H vs. DT) / OPT-821 saponin (G2) was significantly higher than that of IgG (P <0.05).

All publications, patents, and patent applications cited herein are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

It is not intended that the invention be limited to the scope of the embodiments described herein and that these embodiments are illustrative of the individual aspects of the invention and are not intended to limit the scope of the invention. In addition to those described herein, various modifications of the models and methods of the present invention will be apparent to those skilled in the art from the foregoing description and teaching, and they are likewise included within the scope of the present invention. Such variations or other embodiments may be practiced without departing from the true scope and spirit of the invention.

Claims (37)

(a) a carbohydrate antigen or an immunogenic fragment thereof; And
(b) a vaccine comprising a toxoid protein,
Wherein the ratio of carbohydrate antigen to toxoid protein is in the range of 5: 1 to 39: 1. 2. The vaccine of claim 1, wherein the toxoid protein is a diphtheria toxin (DT). 3. The vaccine of claim 2, wherein the ratio of carbohydrate antigen and DT is in the range of 8: 1 to 24: 1. 7. The vaccine of claim 1, wherein the toxoid protein is tetanus toxoid (TT). 5. The vaccine of claim 4, wherein the ratio of carbohydrate antigen and TT ranges from 7: 1 to 12: 1. The vaccine according to claim 1, wherein the carbohydrate antigen is selected from Globo H, SSEA-3, SSEA-4 or Gb-4. 7. The vaccine according to claim 6, wherein the carbohydrate antigen is Globo H. 3. The vaccine according to claim 2, wherein DT is a diphtheria toxin cross-reacting substance or diphtheria toxoid. 9. The vaccine according to claim 8, wherein the diphtheria toxin cross-reacting substance is selected from CRM30, CRM45, CRM 176, CRM 197 or CRM 228. The vaccine according to claim 1, further comprising a saponin adjuvant. 11. The vaccine according to claim 10, wherein the saponin adjuvant is OPT-821 saponin. 3. The vaccine of claim 1, further comprising an alpha -galactosyl-ceramide (alpha-GalCer) adjuvant. 13. The vaccine according to claim 12, wherein the? -GalCer agonist has the following structure:

(a) a carbohydrate antigen or an immunogenic fragment thereof;
(b) a toxoid protein; And
(c) a pharmaceutical composition comprising a pharmaceutically acceptable carrier,
Wherein the ratio of carbohydrate antigen to DT is in the range of 5: 1 to 39: 1. 15. The pharmaceutical composition according to claim 14, wherein the toxoid protein is a diphtheria toxin (DT). A separate compound of formula (I): &lt; EMI ID =

In this formula,
R ¹ is selected from? -D-apios or? -D-xylose;
R &lt; ² &gt; and R &lt; ³ &gt; are H,

. 17. The compound of claim 16, wherein R &lt; ^{1 &gt;} is beta -D-apiose and R &lt; ^{2 &}

&Lt; / RTI &gt; and R &lt; ³ &gt; The method of claim 16, wherein R ¹ is β-D- Bahia OSU and, R ² is H, R ³ is

Lt; / RTI &gt; 17. The compound of claim 16, wherein R &lt; ^{1 &gt;} is beta -D-xylose and R &lt; ^{2 &}

&Lt; / RTI &gt; and R &lt; ³ &gt; The method of claim 16, wherein R ¹ is β-D- xylose and, R ² is H, R ³ is

Lt; / RTI &gt; A pharmaceutical composition comprising a compound of formula (I): or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier,

In this formula,
R ¹ is selected from? -D-apios or? -D-xylose;
R &lt; ² &gt; and R &lt; ³ &gt; are H,

. OPT-821 saponins comprising one or more of the following isolated compounds:
1857 Compound V1A;
1857 Compound V1B;
1857 Compound V2A; And
1857 Compound V2B. 23. The composition of claim 22, further comprising one or more of the following isolated compounds: OPT-821 saponin:
1989 Compound V1A;
1989 Compound V1B;
1989 Compound V2A; And
1989 Compound V2B. About 1 to about 15 mole percent of a 1857 compound mixture; And
OPT-821 saponin comprising about 85 to about 99 mole percent of a mixture of 1989 compounds. 25. The method of claim 24,
About 60-70 mole% of 1857 compound V1A;
About 1-5 mole% of 1857 compound V1B;
About 30-40 mole percent of 1857 compound V2A; And
OPT-821 saponin comprising about 0.1-3 mole% of 1857 compound V2B. 25. The method of claim 24,
About 60-70 mole% of the 1989 compound V1A;
About 1-5 mole% of the 1989 compound V1B;
About 30-40 mole% of the 1989 compound V2A; And
OPT-821 saponin comprising about 0.1-3 mole% of 1989 compound V2B. (a) a carbohydrate antigen or an immunogenic fragment thereof; And
(b) a vaccine comprising OPT-821 saponin. 28. The vaccine of claim 27, wherein the carbohydrate antigen or immunogenic fragment thereof is selected from Globo H, SSEA-3, SSEA-4, Gb-4 or a mixture thereof. 28. The vaccine of claim 27, further comprising a carrier protein. 30. The vaccine of claim 29, wherein the carrier protein is an inactivated diphtheria toxin (DT). 31. The vaccine of claim 30, wherein DT is a diphtheria toxin cross-reacting substance or diphtheria toxoid. 32. The vaccine of claim 31, wherein the diphtheria toxin cross-reacting substance is selected from CRM30, CRM45, CRM 176, CRM 197 or CRM 228. 30. The vaccine of claim 29, wherein the carrier protein is KLH. (a) the vaccine of claim 1 or 27; And
(b) a pharmaceutically acceptable carrier. A method for inhibiting cancer cells, comprising administering an effective amount of the vaccine of claim 1 or 27 to a subject in need thereof to inhibit cancer cells. 36. The method of claim 35, wherein the cancer is a Globo H-expressing cancer. 37. The method of claim 36, wherein the Globo H expressing cancer is breast cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer and endometrial cancer.

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